Part:BBa_K1890002
Silicatein gene, fused to transmembrane domain of OmpA, with strong RBS
Introduction
Silicatein, originating from the demosponge Tethya aurantium, catalyzes the formation of polysilicate. As described by Curnow et al, the silicatein gene was fused to the transmembrane domain of outer membrane protein A (OmpA), in order to display it at the surface of the cell [1][2]. The fusion of silicatein and OmpA is constructed according to Francisco et al, consisting of the transmembrane domain of OmpA together with the signaling peptide and the first nine N-terminal amino acids of lipoprotein (Lpp), both of which are native proteins from Escherichia coli [3]. The coding sequence in this BioBrick is set downstream of strong RBS BBa_B0034.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 192
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Usage and Biology
Silicatein is an enzyme natively found in demosponges and diatoms, where it catalyzes the condensation of silica to form the typical skeletal elements. Here, we use the enzyme to create a polysilicate layer around the host organism E. coli (Figure 1). The gene is fused to the transmembrane domain of OmpA in order to display the protein at the cell membrane. This biobrick was expressed under the control of an inducible promoter (Lac-promoter), to do so it was cloned in a backbone containing the promoter and all machinery necessary for it to work. This backbone was obtained from pBbS5a-RFP, a gift from Jay Keasling (Addgene plasmid # 35283) [4].
Characterization
This biobrick was expressed in E. coli BL21 strain. Cells were grown overnight in selective LB. They were transferred to fresh medium and grown until in exponential phase. Then IPTG was added to induce expression. After a subsequent incubation of three hours, the medium was supplemented with silicic acid as substrate for silicatein. After another three hours, the silicate layer was considered to be formed [7]. A change in structure was observed for these cultures (Figure 2).
In order to characterize the formation of a polysilicate layer around E. coli, we performed multiple experiments.
- Rhodamine 123 staining
- Growth study
- SEM imaging
- TEM imaging
Staining with Rhodamine 123
In this experiment we imaged the silicatein expressing cells with a fluorescence microscope, after treating them with a fluorescent dye. The fluorescent dye Rhodamine 123 (Sigma) has shown to bind specifically to polysilicate [5]. Cells were stained according to the protocol based on Li et al. and Müller et al. [5][6]. Rhodamine was excited with a wavelength of 395 nm.
From figure 3 we can see that the strain transformed with OmpA-silicatein clearly has a different output from the negative control. The fluorescence is only localized at the cells. From this we can conclude the Rhodamine has stained the cells and therefore these cells contain a polysilicate layer.
Viability
Since the silicatein expressing cells are to cover themselves in polysilicate, their nutrient supply might be limited by diffusion, which can eventually result in cell death. To investigate whether this is indeed the case, a growth study was performed (Figure 4).
Cells were grown overnight in selective LB. They were transfered to fresh medium and grown until in exponential phase. Then IPTG was added to induce expression. After a subsequent incubation of three hours, the medium was supplemented with silicic acid as substrate for silicatein. During the following five hours samples were taken, of which a 10-6 dilution was plated on selective LB plates. Colony forming units (cfu) were counted the day after. As a negative control, cells expressing another type of silicatein BBa_K1890002, not supplemented with silicic acid were used.
This figure suggests that either the polysilicate layer inhibits nutrient diffusion into the cell, or the sodium silicate has a detrimental effect on growth.
References
[1] Curnow, P., Kisailus, D., & Morse, D. E. (2006). Biocatalytic synthesis of poly(L-lactide) by native and recombinant forms of the silicatein enzymes. Angewandte Chemie - International Edition, 45(4), 613–616.
[2] Curnow, P., Bessette, P. H., Kisailus, D., Murr, M. M., Daugherty, P. S., & Morse, D. E. (2005). Enzymatic synthesis of layered titanium phosphates at low temperature and neutral pH by cell-surface display of silicatein-?? Journal of the American Chemical Society, 127(45), 15749–15755.
[3] Francisco, J. a, Earhart, C. F., & Georgiou, G. (1992). Transport and anchoring of beta-lactamase to the external surface of Escherichia coli. Proceedings of the National Academy of Sciences of the United States of America, 89(April), 2713–2717.
[4] Lee, T. S., Krupa, R. A., Zhang, F., Hajimorad, M., Holtz, W. J., Prasad, N., … Keasling, J. D. (2011). BglBrick vectors and datasheets: A synthetic biology platform for gene expression. Journal of Biological Engineering, 5, 12. http://doi.org/10.1186/1754-1611-5-12
[5] Li, C. W., Chu, S., & Lee, M. (1989). Characterizing the silica deposition vesicle of diatoms. Protoplasma, 151(2-3), 158–163.
[6] Müller, W. E. G., Rothenberger, M., Boreiko, A., Tremel, W., Reiber, A., & Schröder, H. C. (2005). Formation of siliceous spicules in the marine demosponge Suberites domuncula. Cell and Tissue Research, 321(2), 285–297.
[7] Müller, W. E. G., Engel, S., Wang, X., Wolf, S. E., Tremel, W., Thakur, N. L., … Schröder, H. C. (2008). Bioencapsulation of living bacteria (Escherichia coli) with poly(silicate) after transformation with silicatein-α gene. Biomaterials, 29(7), 771–779. http://doi.org/10.1016/j.biomaterials.2007.10.038
| None |